The art has recognized the potential of nanoparticles, which are generally defined as spherical particles having sizes ranging from approximately a few nanometers up to a few microns, for use in different applications in chemistry, biology and medicine. The preparation of various nanoparticles and uses thereof are described in the following publications: Scientific and Clinical Applications of Magnetic Carriers, Ed. U. Hafeli, W. Schutt, J. Teller and M. Zborowski, Plenum Press, N.Y. 37-51, 53-67 and 135-148 (1997); Recent Res. Developments in Polymerization Science, Ed. S. G. Patidalai, Transworld Research Network 1, 51-78 (1997); O. Melamed and S. Margel, J. of Colloid and Interface Science 241, 357 (2001); V. Bendikiene and B. Juodka, Stability and Stabilization of Biocatalysts, Ed. A. Ballesteros, F. J. Plou, J. L. Iborra and P. J. Halling, Elsevier Science 583-588 (1998) and J. Parrado and J. Bautista, Biosci Biotech. Biochem. 59 (5), 906 (1995).
The blood clotting process involves the cleavage of the plasma highly soluble molecule fibrinogen by the action of the proteolytic enzyme thrombin, following which the monomers obtained associate together to form fibrin, in the form of long, insoluble fibers. The formation of the insoluble fibrin matrix may be accelerated by the presence of factor XIII and/or the presence of Ca+2 ions. Thrombin converts factor XIII to factor XIIIa, which, in the presence of CaCl2, crosslinks the fibrin matrix to give a highly crosslinked polymer.
The art proposed various formulations, known as fibrin glues, or fibrin sealants, in order to allow effective and rapid interaction between fibrinogen and thrombin, and devices useful for delivering said formulations to the bleeding site, thereby mimicking the final step of blood clotting process resulting in the production of the desired fibrin clot.
For example, the commercially available product Tisseel® Fibrin Sealant (Immuno AG, Austria) is based on a two compartments syringe for separately holding a thrombin solution and fibrinogen solution. In use, the two components are simultaneously mixed and applied on the bleeding area to form the insoluble fibrin glue matrix.
Another commercially available product, Quixil® (Omrix Biopharmaceuticals, Belgium) is based on one-compartment syringe, or similar arrangement, composed of a thrombin solution only. The thrombin solution may also contain other components, e.g. antifibrinolytic agents such as aprotinin or tranexamic acid and/or factor XIII and/or CaCl2. The thrombin solution is then sprayed, or compressed, or applied, on the bleeding area to form the insoluble fibrin by interacting with the fibrinogen content of the blood.
TachoComb® (NYCOMED GmbH, Munchen), is prepared by covering a sheet of collagen with human fibrinogen and bovine thrombin. Aprotinin is added to prevent early degradation of the fibrin clot by plasmin. For this purpose, the solid components: fibrinogen, thrombin and aprotinin are dispersed in an organic medium, and the suspension is applied on a sheet of collagen. The organic medium is evaporated, leaving a dried layer of the components of fibrin glue adsorbed on the collagen surface. When the coating comes in contact with blood or other liquids, the components dissolve and fibrin is formed.
A. Sugitachi et al. [Progress in Artificial Organs, Ed. Y. Nose, C. Kjellstrand and P. Ivanovich, 1020-1022 (1985)] describe dry fine flakes containing thrombin, gelatin and factor XIII, suitable for treating hemostasis.
Horak et al. [Polymers in Medicine 21 (1-2), 31 (1991)] describe a thrombin-containing hydrogel, based on poly(2-hydroxyethyl methacrylate), that is useful for endovascular occlusion.
Liu et al. [Analytical Biochemistry 147, 49 (1985)], describe thrombin-gold nanoparticles, that are prepared by physical adsorption of a monolayer of thrombin onto gold nanoparticles of 16.5±1.8 nm diameter.
It is a purpose of the present invention to provide a novel thrombin formulation and therapeutic compositions based thereon, that may be used in the preparation of a fibrin sealant.
It is another purpose of the invention to provide a thrombin formulation that is highly stable and easily deliverable, and a process for preparing the same.
It is a further purpose of the invention to provide a thrombin formulation that permits rapid formation of the fibrin clot.
Further objects and advantages of the present invention will become apparent as the description proceeds.